Crystallographic Snapshots of Sulfur Insertion by Lipoyl Synthase

Crystallographic snapshots of sulfur insertion by lipoyl synthase Martin I. McLaughlina,b,1, Nicholas D. Lanzc, Peter J. Goldmana, Kyung-Hoon Leeb, Squire J. Bookerb,c,d, and Catherine L. Drennana,e,f,2 aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802; cDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802; dHoward Hughes Medical Institute, The Pennsylvania State University, University Park, PA 16802; eDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and fHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, and approved July 5, 2016 (received for review March 8, 2016) Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms substrate and at an intermediate stage in the reaction, just after at the unactivated C6 and C8 positions of a protein-bound octanoyl insertion of the C6 sulfur atom but before sulfur insertion at C8. chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethio- Results nine (AdoMet) radical chemistry; the remainder of the reaction The crystal structure of LipA from M. tuberculosis was de- mechanism, especially the source of the sulfur, has been less clear. termined to 1.64-Å resolution by iron multiwavelength anoma- One controversial proposal involves the removal of sulfur from a lous dispersion phasing (Table S1). The overall fold of LipA consists second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in de- of a (β/α)6 partial barrel common to most AdoMet radical enzymes struction of the cluster during each round of catalysis. Here, we (14), an N-terminal α-helical extension, and a C-terminal ex- present two high-resolution crystal structures of LipA from Myco- tension (Fig. 1A and Fig. S1). The [4Fe-4S] cluster that binds bacterium tuberculosis: one in its resting state and one at an in- AdoMet and is responsible for radical generation is coordinated termediate state during turnover. In the resting state, an auxiliary by three cysteines of the CX3CXΦC motif (Φ denotes an aro- [4Fe-4S] cluster has an unusual serine ligation to one of the irons. matic residue) that is on the AdoMet radical loop between β1 After reaction with an octanoyllysine-containing 8-mer peptide and α1 of the barrel (Fig. S2). The second (auxiliary) [4Fe-4S] substrate and 1 eq AdoMet, conditions that allow for the first sulfur cluster is sandwiched between the N- and C-terminal extensions insertion but not the second insertion, the serine ligand dissociates (Fig. 1A). As also observed in a recent structure of LipA from from the cluster, the iron ion is lost, and a sulfur atom that is still Thermosynechococcus elongatus (15), three of the iron ions of the part of the cluster becomes covalently attached to C6 of the octanoyl auxiliary cluster are ligated by cysteines residing in a conserved substrate. This intermediate structure provides a clear picture of N-terminal CX CX C motif, whereas the fourth iron ion is li- – 4 5 iron sulfur cluster destruction in action, supporting the role of the gated by S292 found in a conserved C-terminal R(S/T)SΦ motif auxiliary cluster as the sulfur source in the LipA reaction and de- (Fig. 1B and Fig. S1). R290 of this R(S/T)SΦ motif extends scribing a radical strategy for sulfur incorporation into completely between the two [4Fe-4S] clusters and covers the most exposed unactivated substrates. face of the auxiliary cluster with the aliphatic portion of its side iron–sulfur cluster | radical SAM enzyme | lipoic acid Significance he functionalization of aliphatic carbon centers is widely Lipoic acid, an enzyme cofactor in central metabolism and a regarded as one of the most kinetically challenging reactions T livestock feed supplement, is produced on an industrial scale by in nature, a striking example of which is found in the biosynthesis a costly multistep synthesis. Nature makes lipoic acid in one of the lipoyl cofactor, famous for its central role as the “swinging ” step by the chemically challenging addition of two sulfur arm of the pyruvate dehydrogenase enzyme complex. Lipoyl atoms to an inert fatty acid chain. The sulfur source in this synthase (LipA) generates the lipoyl cofactor by insertion of two reaction has been controversial, and its identity has implica- sulfur atoms at C6 and C8 of a protein-bound n-octanoyl chain, – tions for engineering microorganisms to overproduce lipoic sites distal from the nearest functionality (1 4). LipA and the acid. Structural characterization of a lipoyl synthase enzyme closely related biotin synthase (BioB) (Scheme 1) are founding captured in the middle of catalysis shows unequivocally that members of the ever-expanding S-adenosyl-L-methionine (Ado- the enzyme obtains its sulfur atoms by cannibalizing an iron– Met) radical enzyme superfamily that uses a [4Fe-4S] cluster to sulfur cluster, another ancient and essential cofactor. This re- reductively cleave the C5′-S bond of AdoMet, generating me- sult reveals an alternative strategy for sulfur mobilization and thionine and a 5′-deoxyadenosyl radical (5′-dA•), a powerful an unexpected self-sacrificial role for iron–sulfur clusters oxidant (5, 6). LipA requires 2 eq AdoMet—one per sulfur in- in biology. sertion—and two sulfur atoms to produce 1 eq lipoyl product through radical-based chemistry (4). One of the most contro- Author contributions: M.I.M., N.D.L., S.J.B., and C.L.D. designed research; M.I.M., N.D.L., versial aspects of the LipA and BioB reactions is the source of P.J.G., and K.-H.L. performed research; N.D.L. and K.-H.L. contributed new reagents/ analytic tools; M.I.M., N.D.L., P.J.G., S.J.B., and C.L.D. analyzed data; and M.I.M., N.D.L., the sulfur. AdoMet radical enzymes that catalyze sulfur insertion P.J.G., S.J.B., and C.L.D. wrote the paper. – always seem to have an additional iron sulfur (Fe/S) cluster: a The authors declare no conflict of interest. [2Fe-2S] cluster in BioB (7), a [4Fe-4S] cluster in LipA (8), and a This article is a PNAS Direct Submission. [4Fe-4S] cluster in the methylthiotransferases RimO and MiaB Data deposition: The atomic coordinates and structure factors have been deposited in the (9, 10). These auxiliary clusters in LipA and BioB have been pro- Protein Data Bank, www.wwpdb.org (PDB ID codes 5EXI, 5EXJ, and 5EXK). posed to be cannibalized during turnover to supply the inserted 1Present address: Department of Chemistry, University of Illinois at Urbana–Champaign, sulfur atom(s) (8, 11–13), a proposal that has not enjoyed universal Urbana, IL 61801. agreement. Here, we investigate this incredible sulfur insertion 2To whom correspondence should be addressed. Email: [email protected]. chemistry through crystallographic snapshots of LipA from the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. human pathogen Mycobacterium tuberculosis both in the absence of 1073/pnas.1602486113/-/DCSupplemental. 9446–9450 | PNAS | August 23, 2016 | vol. 113 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1602486113 Downloaded by guest on September 30, 2021 with the abstraction of the pro-R H• from C6 (2) and inversion of configuration on sulfur insertion (Fig. 2B and Fig. S4). Consistent with the first half reaction having taken place, we find that the substrate octanoyl group is covalently attached to the auxiliary cluster by a bond between C6 and a cluster sulfur atom (Fig. 1D). S292 is no longer a ligand to the cluster, and the iron atom that was coordinated by S292 in the substrate-free structure is no longer present (Fig. 2C, Inset). Previously, a Mössbauer spec- troscopic study revealed the loss of an iron atom from the auxiliary cluster on the timescale of the first sulfur insertion (16), and here, we see which iron is lost, and potentially, how it is lost: a solvent- filled cavity exists on one side of the cluster that extends to the protein surface (Fig. 2C). These structural data also present an Scheme 1. The reactions catalyzed by the AdoMet radical sulfur insertion explanation of why iron loss could be of mechanistic significance. enzymes (Upper)BioBand(Lower) LipA. The inserted sulfur atoms are The presence of this iron atom would block access of the two closest marked in red. sulfur atoms of the auxiliary cluster (5.5 and 5.3 Å) to C8 of the octanoyl moiety (Fig. S4), hindering the second sulfur insertion. chain (Fig. 1B).Intheabsenceofsubstrate,theactivesiteis Discussion open and filled with solvent, with a 15.3-Å distance between the The chemical difficulty of synthesizing the lipoyl and biotin co- two clusters (Fig. 2A). Thus, for the auxiliary cluster to serve as factors cannot be overstated. This point is exemplified by the fact the source of sulfur, displacement of R290 and a conformational that 10–30 tons of biotin sold each year are produced by a chemical tightening of the active site seem to be required. synthetic route that is estimated to have as many as 15 steps (18). To investigate the mechanism of sulfur insertion by LipA, we First, the challenge of regio- and stereoselective C–Hbondacti- crystallographically characterized a stable but kinetically compe- vation must be overcome, and second, a route for sulfur func- tent reaction intermediate that was previously described by Lanz tionalization must be achieved. Here, we find that the LipA et al. (16) for Escherichia coli LipA.

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